1. Field of the Invention
The field of the invention relates to plants and plant genes, including both plant mutants and transgenic plants containing a gene that confers an ethylene insensitive phenotype. Also encompassed by the invention are methods of using the disclosed plant gene to confer an ethylene insensitive phenotype.
2. Description of the Related Art
Ethylene is an endogenous plant hormone that affects many aspects of growth and development, such as germination, flower and leaf senescence, fruit ripening, leaf abscission, cell fate determination in root epidermis, root nodulation, sex determination, programmed cell death, and responsiveness to stress and pathogen attack (Abeles et al., 1992; Johnson and Ecker, 1998). The biosynthetic pathway for this hormone is now well-established. First, the amino acid methionine is converted into ethylene via S-adenosylmethionine (“SAM” or “AdoMet”) and 1-aminocyclopropane-1-carboxylic acid (ACC) (Yang and Hoffman, 1984). The key enzymes of ethylene biosynthesis, AdoMet synthase, ACC synthase and ACC oxidase, have now been cloned and characterized (Johnson and Ecker, 1998; Morgan P W, 1997).
Ethylene is involved in regulating many physiological processes. Examples, include responses to pathogens, initiation of fruit ripening, cell wall formation/degradation, leaf epinasty (downward curvature of leaf), inhibition of seedling elongation or seed germination, and the promotion (or inhibition, in some species) of flowering. Ethylene also regulates the abscission of plant organs such as leaves, fruits, and flowers (see, e.g., Taiz and Zeiger, 1991, Plant Physiology, Benjamin/Cummings Publishing Company, Inc., p. 474–482). Therefore, plants having a decreased sensitivity to ethylene may have several agricultural uses. For example, plants having a decreased sensitivity to ethylene may have better storage characteristics. Fruits of such plants may ripen more slowly. This could be an advantage for post-harvest handling of agricultural products, such as processing, packaging, and storage of fruit. There may be less of a loss of fruit crops due to such traditionally damaging problems as rotting, over-ripening, and degradation. It may be possible to better control the ripening rate and ripening characteristics of plants carrying these modified EDF genes. It is possible, as well, to link the modified genes to specific promoters in order to better modulate the expression of the genes so that the response to ethylene is turned off at certain times or in certain tissues, while acting normally in other parts of the plant or at other times in development.
Plants respond to ethylene through a family of integral membrane receptors. In Arabidopsis, at least five family members are involved, including: ETHYLENE RECEPTOR1 (ETR1), ETR2, ETHYLENE INSENSITIVE4 (EIN4), ETHYLENE RESPONSE SENSOR1 (ERS1), and ERS2 (Chang et al., 1993; Hua et al., 1995; Hua et al., 1998; Sakai et al., 1998). Ethylene binds to the receptors via a copper cofactor (Rodriguez et al., 1999) and genetic studies suggest that hormone binding inactivates the receptors (Hua and Meyerowitz, 1998). In the absence of ethylene, the receptor are predicted to be functionally active histidine kinases which activate a Raf-like S/T kinase, CONSTITUTIVE TRIPLE RESPONSE1 (CTR1), also a negative regulator of the pathway (Kieber et al., 1993). Genetic studies also predict that EIN2, EIN3, EIN5, and EIN6 (Roman et al., 1995) are positive regulators of the ethylene response.
EIN2 is a metal ion transporter-related integral-membrane protein, whose function is not well-understood (Alonso et al., 1999). The nuclear protein EIN3 and its paralogs, the EIN3-LIKE proteins (EILs), are transcription factors that bind to the promoters of ethylene-response genes such as ETHYLENE RESPONSE FACTOR 1 (ERF1) and initiate a transcriptional cascade leading to the regulation of ethylene target genes (Chao et al., 1997; Solano et al., 1998).
Ethylene and Disease Resistance
Ethylene gas is released upon pathogen infection and is thought to be a part of the plant defense mechanism against the spread of the pathogen. In the past year, several studies have demonstrated that a functional ethylene signaling pathway is required for resistance against some, but not all, pathogens. EIN2 was shown to be essential for pathogen-mediated systemic induction of the basic chitinase PR-3 and a hevein-like gene PR-4 in Arabidopsis upon infection with the fungus Altemaria brassicicola (Thomma et al., 1999). Local induction of the HEL, CHIB and PDF1.2 genes by a culture filtrate from the virulent Gram-negative bacterium Erwinia carotovora subsp. carotovora was also severely reduced in the ein2-1 and etr1-1 mutants (Norman-Setterblad et al., 2000). Furthermore, ein2-1 plants exhibited greater susceptibility to infections by E. carotovora subsp. carotovora (Norman-Setterblad et al., 2000) and the fungus Botrytis cinerea, but not to infection by avirulent strains of the fungi A. brassicicola and Peronospora parasitica (Thomma et al., 1999).
Ethylene Response in Animals
The possession of an ethylene signal transduction pathway is not unique to Arabidopsis. Orthologs of all the major signaling components known to be involved in this Arabidopsis pathway have been identified in several other plant species (Chang and Shockey, 1999; Johnson and Ecker, 1998) (Ecker, unpublished). Moreover, a bacterial protein that has both sequence homology to the transmembrane domain of ETR1 and ethylene binding properties has been isolated from Synechocystis sp. (Rodriguez et al., 1999). Recently, an animal species, Suberites domuncula, has been shown for the first time to respond to ethylene, both physiologically and at the molecular level (Krasko et al., 1999). In this sponge, ethylene can repress starvation-induced apoptotic cell death, and the mRNA levels of at least two genes, SDERR and CaM kinase II, are up-regulated as a result of ethylene exposure (Krasko et al., 1999). Although it is not yet clear whether this animal can sense and respond to ethylene gas via a conventional ‘plant-specific’ pathway, the fact that gene expression is affected suggests the existence of some sort of perception and transduction pathway for this gas signal.
Thus, what is needed in the art are plants with altered ethylene sensitivity in order to provide more of the effects listed above.